Three-phase partitioning (TPP) is a bioseparation technique used for the extraction, concentration and purification of biomolecules. The technique consists of simultaneous addition of a salt (generally ammonium sulfate) to the crude extract followed by the addition of an organic solvent (generally t-butanol). In the present study, alpha-galactosidase was purified from pepino (Solanum muricatum) fruit by TPP. The influence of various process parameters (ammonium sulfate saturation, crude extract to t-butanol ratio and pH) on alpha-galactosidase partitioning is investigated to get highest purity fold and yield. The results showed that, 50% (w/v) ammonium sulfate saturation with 1:1.5 crude extract to t-butanol ratio at pH 5.25 gave 6.2-fold purification with 127% activity recovery of alpha-galactosidase. Characterization and determination of biochemical properties of the partitioned alpha-galactosidase were also aimed. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed considerable purification and the molecular weight of the enzyme was nearly found to be as 38 kDa. Various parameters (temperature, pH and substrate concentration) affecting to the enzyme activity and stability were studied. The optimum temperature and pH of the partitioned alpha-galactosidase were found to be 50 degrees C and pH 5.5, respectively. The enzyme was very stable at the temperature ranged from 37 to 45 degrees C and also in a pH range of 4.5-7.0. The kinetic constants; K(m) and V(max) were determined to be 0.37 mM and 0.46 U, respectively. The results indicated that, TPP technique is very attractive process for purification of pepino alpha-galactosidase and the characteristic properties of the enzyme partitioned by TPP make the enzyme good candidates for its several industrial applications. (C) 2011 Elsevier B.V. All rights reserved.